Urinary bladder cancer (UBC) patients at muscle invasive stage have poor clinical outcome, due to high propensity for chemoresistance. Cancer-associated fibroblasts (CAFs), one of the principal constituents of the tumor stroma, play an important role in tumor development. Exosomal microRNAs (miRNAs) are emerging mediators of cancer-host crosstalk with other cells around. However, it is unclear whether CAFs from UBC promote cancer chemoresistance through exosomal miRNAs.

METHODS

We obtained human bladder cancer specimens and adjacent normal tissues from Nanjing Drum Tower Hospital. The bladder cancer specimen used for isolation of stromal fibroblasts was diagnosed as muscle invasive bladder cancer with histological grade II. Bladder cancer cells (T24 and 5637) were treated with the conditional medium (CM) or exosomes from normal fibroblasts (NFs) and CAFs, as well as an equal volume of complete culture medium. Exosomes were isolated from NF-CM or CAF-CM using ultracentrifugation or Total Exosome Isolation kit. T24 cells and NFs or CAFs in 0.1 ml 50% Matrigel were injected subcutaneously into the right flank of male athymic nude mice. Gemcitabine (40 mg/kg body weight) and cisplatin (6 mg/kg body weight) were respectively given on day 1, 5, 8 ,11 and day 2 via intraperitoneal injection. Tumor diameters were measured with calipers every 3 days and tumor volumes were calculated. The log-rank t-test was used for survival analysis. P value less than 0.05 was considered statistically significant.

RESULTS

We found that co-culture with UBC CAFs promoted T24 xenograft tumor stemness in vivo. Compared to NFs, CAF cells promotes bladder cancer cell stemness via secreted exosomes in vitro. We conducted microarray assay to identify exosomal miRNAs potentially secreted by UBC CAFs and found that miR-146a was the most highly expressed and secreted. Mimic miR-146a also enhanced the stemness of UBC cells. To further confirm the effect of miR-146a on crosstalk between CAFs and UBC in vivo, we stably overexpressed miR-146a or control vector in NFs (NF/miR-146a and NF/vec) and co-cultured with T24 cells, respectively. CAF or NF/miR-146a significantly enhanced T24 xenograft tumor stemness and chemoresistance. Bioinformatic analyses found that AMPKα contain potential miR-146a binding sites in its 3&[prime]-UTRs. We further comfirmed that exosomal miR-146a repress AMPKα to induce mTOR signal pathway. Consistently, serum exosomal miR-146a expression is positively correlated with recurrence, and patients with high miR-146a had a poor prognosis.